Crystal structure of the plant dual-affinity nitrate transporter NRT1.1

Abstract

Nitrate is a primary nutrient for plant growth, but its levels in soil can fluctuate by several orders of magnitude. Previous studies have identified Arabidopsis NRT1.1 as a dual-affinity nitrate transporter, which can take up nitrate over a wide range of concentrations. The mode of action of NRT1.1 is controlled by phosphorylation of a key residue, Thr101. Yet how this posttranslational modification switches the transporter between two affinity states remains unclear. Here we report the crystal structure of unphosphorylated NRT1.1, which reveals an unexpected homodimer in the inward-facing conformation. In this low-affinity state, the Thr101 phosphorylation site is embedded in a pocket immediately adjacent to the dimer interface, linking the phosphorylation status of the transporter to its oligomeric state. Using a cell-based fluorescence resonance energy transfer assay, we show that functional NRT1.1 indeed dimerizes in the cell membrane and the phosphomimetic mutation of Thr101 converts the protein into a monophasic high affinity transporter by structurally decoupling the dimer. Together with analyses of the substrate transport tunnel, our results establish a phosphorylation-controlled dimerization switch that allows NRT1.1 to uptake nitrate with two distinct affinity modes.

Phosphorylation at serine 52 and 635 does not alter the transport properties of glucosinolate transporter AtGTR1

“…Furthermore, this site is found in the highly conserved EXXE[R/K] motif and shown to be buried in the membrane segment of the close homolog NRT1.1 where it is facing the substrate tunnel and unlikely to be accessible for kinases. 30 This was confirmed by a SWISS-MODEL generated AtGTR2 homology-model where residue Thr58 is situated in the middle of helix 1 (data not shown). IMembrane predicted AtGTR1-S52 and AtGTR1-S635 to be situated in the non-membrane segment of the protein (Fig.…”

NRT1.5/NPF7.3 Functions as a Proton-Coupled H+/K+ Antiporter for K+ Loading into the Xylem in Arabidopsis

“…These four charged residues are highly conserved in most Arabidopsis NRT1 members. However, NRT1.5 and NRT1.8 have simultaneously evolved into noncharged residues in these four sites, suggesting that NRT1.5 and NRT1.8 must have an alternative proton-coupling mechanism compared with other NRT1 members (Sun et al, 2014). This independent evolution of NRT1.5 and NRT1.8 may endow them with the substrate specificity for K + as well as the unique antiport function.…”

“…The substrate specificity of NRT1/PTR transporters is mainly determined by the specific side chains that line the aqueous pocket at the center of the membrane (Parker and Newstead, 2014;Sun et al, 2014;Yan, 2015). However, how plant NRT1/PTR transporters are able to recognize and transport diverse substrates remains unknown.…”

“…Since the cytoplasmic K + concentrations in root parenchyma cells are over dozens of millimolars, NRT1.5 should be a low-affinity H + /K + antiporter. According to the crystal structure of NRT1.1, the "EXXER" motif (containing three conserved residues) on TMH1 together with the conserved residue Lys-164 on TMH4 is responsible for proton coupling (Doki et al, 2013;Parker and Newstead, 2014;Sun et al, 2014). These four charged residues are highly conserved in most Arabidopsis NRT1 members.…”

Reducing the Genetic Redundancy of Arabidopsis PHOSPHATE TRANSPORTER1 Transporters to Study Phosphate Uptake and Signaling

“…Alternatively, some PHT1 members could present a dual affinity, a view put forth in this study for Arabidopsis. A characteristic already identified for other transporters such as the K Cup1/Arabidopsis K + Transporter1 (Fu and Luan, 1998) and the nitrate NRT1;1 (Parker and Newstead, 2014;Sun et al, 2014). The posttranslational modifications for PHT1 involved in such mechanism remain to be identified.…”