Dataset Information

Purine substrate recognition by the nucleobase-ascorbate transporter signature motif in the YgfO xanthine permease: ASN-325 binds and ALA-323 senses substrate.

ABSTRACT: The nucleobase-ascorbate transporter (NAT) signature motif is a conserved 11-amino acid sequence of the ubiquitous NAT/NCS2 family, essential for function and selectivity of both a bacterial (YgfO) and a fungal (UapA) purine-transporting homolog. We examined the role of NAT motif in more detail, using Cys-scanning and site-directed alkylation analysis of the YgfO xanthine permease of Escherichia coli. Analysis of single-Cys mutants in the sequence 315-339 for sensitivity to inactivation by 2-sulfonatoethyl methanethiosulfonate (MTSES(-)) and N-ethylmaleimide (NEM) showed a similar pattern: highly sensitive mutants clustering at the motif sequence (323-329) and a short alpha-helical face downstream (332, 333, 336). In the presence of substrate, N325C is protected from alkylation with either MTSES(-) or NEM, whereas sensitivity of A323C to inactivation by NEM is enhanced, shifting IC(50) from 34 to 14 microM. Alkylation or sensitivity of the other mutants is unaffected by substrate; the lack of an effect on Q324C is attributed to gross inability of this mutant for high affinity binding. Site-directed mutants G333R and S336N at the alpha-helical face downstream the motif display specific changes in ligand recognition relative to wild type; G333R allows binding of 7-methyl and 8-methylxanthine, whereas S336N disrupts affinity for 6-thioxanthine. Finally, all assayable motif-mutants are highly accessible to MTSES(-) from the periplasmic side. The data suggest that the NAT motif region lines the solvent- and substrate-accessible inner cavity, Asn-325 is at the binding site, Ala-323 responds to binding with a specific conformational shift, and Gly-333 and Ser-336 form part of the purine permeation pathway.

SUBMITTER: Georgopoulou E

PROVIDER: S-EPMC2885222 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Purine substrate recognition by the nucleobase-ascorbate transporter signature motif in the YgfO xanthine permease: ASN-325 binds and ALA-323 senses substrate.

Georgopoulou Ekaterini E Mermelekas George G Karena Ekaterini E Frillingos Stathis S

The Journal of biological chemistry 20100420 25

The nucleobase-ascorbate transporter (NAT) signature motif is a conserved 11-amino acid sequence of the ubiquitous NAT/NCS2 family, essential for function and selectivity of both a bacterial (YgfO) and a fungal (UapA) purine-transporting homolog. We examined the role of NAT motif in more detail, using Cys-scanning and site-directed alkylation analysis of the YgfO xanthine permease of Escherichia coli. Analysis of single-Cys mutants in the sequence 315-339 for sensitivity to inactivation by 2-sul ...[more]

PMID: 20406814

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Project description:Gene mutations conferring herbicide resistance may cause pleiotropic effects on plant fitness. Knowledge of these effects is important for managing the evolution of herbicide-resistant weeds. An Echinochloa crus-galli population resistant to acetolactate synthase (ALS) herbicides was collected in a maize field in north-eastern Italy and the cross-resistance pattern, resistance mechanism and fitness costs associated to mutant-resistant plants under field conditions in the presence or absence of intra-specific competition were determined. The study reports for the first time the Ala-122-Asn amino-acid change in the ALS gene that confers high levels of cross-resistance to all ALS inhibitors tested. Results of 3-year growth analysis showed that mutant resistant E. crus-galli plants had a delayed development in comparison with susceptible plants and this was registered in both competitive (3, 7, and 20 plants m-2) and non-competitive (spaced plants) situations. The number of panicles produced by resistant plants was also lower (about 40% fewer panicles) than susceptible plants under no-intraspecific competition. Instead, with the increasing competition level, the difference in panicle production at harvest time decreased until it became negligible at 20 plants m-2. Evaluation of total dry biomass as well as biomass allocation in vegetative parts did not highlight any difference between resistant and susceptible plants. Instead, panicle dry weight was higher in susceptible plants indicating that they allocated more biomass than resistant ones to the reproductive organs, especially in no-competition and in competition situations at lower plant densities. The different fitness between resistant and susceptible phenotypes suggests that keeping the infestation density as low as possible can increase the reproduction success of the susceptible phenotype and therefore contribute to lowering the ratio between resistant and susceptible alleles. If adequately embedded in a medium or long-term integrated weed management strategy, the presence of R plants with a fitness penalty provides an opportunity to minimize or reverse herbicide resistance evolution through the implementation of integrated weed management, i.e., all possible control tools available.

Project description:IntroductionCadmium (Cd) is one of the most detrimental heavy metal pollutants, seriously affecting crop production and human health. Nucleobase-ascorbic acid transporters (NAT) are widely present in many living organisms including plants, animals and microbes; however, the role of NAT in plant Cd tolerance remains unknown.ObjectivesTo identify Cd-induced miRNAs that target HvNAT2 and to determine the role of this gene and its product in Cd tolerance.MethodsHigh-throughput-sequencing was used to identify the miRNA expression profile of barley roots in response to Cd stress. Overexpression (OX) and RNAi lines were then constructed for HvNAT2 and comparative transcriptomic analysis was performed to determine the function of this transporter examining its effects on traits such as Cd uptake/flux and translocation, morphology and antioxidant capacity in relation to Cd tolerance. In addition, phylogenetic analysis was performed to obtain insights into the evolution of HvNAT2.ResultsCd stress-induced genome-wide expression profiles of miRNAs identified a Cd-induced miRNA, miR156g-3p_3, that had HvNAT2 as its target. HvNAT2 was negatively regulated in the high-Cd-accumulating and Cd-tolerant genotype Zhenong8. Evolutionary analysis indicated that orthologues of the plasma membrane localized, HvNAT2, can be traced back to the sister group of land plants, the streptophyte algae. Overexpression of HvNAT2 increases Cd tolerance with higher tissue Cd accumulation but less oxidative damage in transgenic barley plants. RNAi of HvNAT2 leads to a significant reduction of Cd tolerance. The higher Cd accumulation in roots of the OX3 line was also demonstrated by confocal microscopy and electrophysiology. Transcriptome analysis showed that the enhancement of antioxidant capacity by HvNAT2 was related to stress signaling pathways. Furthermore, oxidative stress tolerance in HvNAT2-OX plants was regulated by the synthesis of phytochelatins and the glutathione metabolism cycle.ConclusionOur study reveals a key molecular mechanism of NAT in Cd tolerance in plants that is useful for sustainable agricultural production and management of hazardous this heavy metal for better environment management and ecosystem function.

Dataset Information

Purine substrate recognition by the nucleobase-ascorbate transporter signature motif in the YgfO xanthine permease: ASN-325 binds and ALA-323 senses substrate.

Publications

Purine substrate recognition by the nucleobase-ascorbate transporter signature motif in the YgfO xanthine permease: ASN-325 binds and ALA-323 senses substrate.

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