Biochemical characterization and structure–function relationship of two plant NCS2 proteins, the nucleobase transporters NAT3 and NAT12 from Arabidopsis thaliana

Abstract

Nucleobase ascorbate transporters (NATs), also known as Nucleobase:Cation-Symporter 2 (NCS2) proteins, belong to an evolutionary widespread family of transport proteins with members in nearly all domains of life. We present the biochemical characterization of two NAT proteins, NAT3 and NAT12 from Arabidopsis thaliana after their heterologous expression in Escherichia coli UraA knockout mutants. Both proteins were shown to transport adenine, guanine and uracil with high affinities. The apparent KM values were determined with 10.12μM, 4.85μM and 19.95μM, respectively for NAT3 and 1.74μM, 2.44μM and 29.83μM, respectively for NAT12. Competition studies with the three substrates suggest hypoxanthine as a further substrate of both transporters. Furthermore, the transport of nucleobases was markedly inhibited by low concentrations of a proton uncoupler indicating that NAT3 and NAT12 act as proton-nucleobase symporters. Transient expression studies of NAT-GFP fusion constructs revealed a localization of both proteins in the plasma membrane. Based on the structural information of the uracil permease UraA from E. coli, a three-dimensional experimentally validated homology model of NAT12 was created. The NAT12 structural model is composed of 14 TM segments and divided into two inverted repeats of TM1-7 and TM8-14. Docking studies and mutational analyses identified residues involved in NAT12 nucleobase binding including Ser-247, Phe-248, Asp-461, Thr-507 and Thr-508. This is the first study to provide insight into the structure-function of plant NAT proteins, which reveals differences from the other members of the NCS2 protein family.

Insight on specificity of uracil permeases of the NAT/NCS2 family from analysis of the transporter encoded in the pyrimidine utilization operon of Escherichia coli

“…In XanQ, replacement of the corresponding Phe94 with Cys results in marginal activity but replacement with Ile or Tyr yields highly active mutants which display reduced affinity for xanthine and impaired affinity for 3‐methylxanthine (F94I) or 2‐thioxanthine and 6‐thioxanthine (F94Y) (Karena and Frillingos, ). Of the homologs from higher eukaryotes, in the Arabidopsis thaliana AtNAT12, a high‐affinity transporter for adenine, guanine and uracil, replacement of the corresponding Phe248 with Ala was found to retain 20–35% of activity with all substrates (Niopek‐Witz et al ., ). Overall, the phenyl group at Phe73/155 plays a critical but not irreplaceable role in substrate coordination which seems to be retained throughout the family, except in RutG and all enterobacterial RutG orthologs, which have Ala at this position (Fig.…”

Apoplastic Nucleoside Accumulation in Arabidopsis Leads to Reduced Photosynthetic Performance and Increased Susceptibility Against Botrytis cinerea

“…As inosine cannot be converted to xanthosine directly (Dahncke and Witte, 2013 ), it first has to become deribosylated to hypoxanthine either in the apoplast by NSH3 or after import via ENT3 by NSH1 to become further catabolized to xanthine by xanthine dehydrogenase (Hesberg et al, 2004 ). Apoplastic hypoxanthine can subsequently be imported by plasma membrane located NAT3 and NAT12 transport proteins (Niopek-Witz et al, 2014 ). When direct import and extracellular catabolism are not active in ent3:nsh3 mutants, inosine cannot function as efficient nitrogen source.…”

Nucleotide Metabolism in Plants

“…However, one should note that definitive evidence for a function of PLUTO in uracil transport into plastids in vivo has not yet been presented for any plant. Other transporters capable of uracil transport have been identified, but they are located in the plasma membrane (Schmidt et al, 2004;Niopek-Witz et al, 2014).…”