Characterization of three novel members of the Arabidopsis thaliana equilibrative nucleoside transporter (ENT) family

Abstract

Research on metabolism of nucleotides and their derivatives has gained increasing interest in the recent past. This includes de novo synthesis, analysis of salvage pathways, breakdown and transport of nucleotides, nucleosides and nucleobases. To perform a further step towards the analysis of nucleoside transport in Arabidopsis, we incubated leaf discs with various radioactively labelled nucleosides. Leaf cells imported labelled nucleosides and incorporated these compounds into RNA, but not into DNA. Furthermore, we report on the biochemical properties of three so far uncharacterized members of the Arabidopsis ENT (equilibrative nucleoside transporter) family (AtENT4, AtENT6 and AtENT7). After heterologous expression in yeast, all three proteins exhibited broad substrate specificity and transported the purine nucleosides adenosine and guanosine, as well as the pyrimidine nucleosides cytidine and uridine. The apparent K(m) values were in the range 3-94 microM, and transport was inhibited most strongly by deoxynucleosides, and to a smaller extent by nucleobases. Typical inhibitors of mammalian ENT proteins, such as dilazep and NBMPR (nitrobenzylmercaptopurine ribonucleoside, also known as nitrobenzylthioinosine) surprisingly exerted almost no effect on Arabidopsis ENT proteins. Transport mediated by the AtENT isoforms differed in pH-dependency, e.g. AtENT7 was not affected by changes in pH, AtENT3, 4 and 6 exhibited a less pronounced pH-dependency, and AtENT1 activity was clearly pH-dependent. Using a GFP (green fluorescent protein)-fusion protein transiently expressed in tobacco leaf protoplasts, a localization of AtENT6 in the plant plasma membrane has been revealed.

Uridine-Ribohydrolase Is a Key Regulator in the Uridine Degradation Pathway of Arabidopsis

“…The measured maximal activities for UK and UPRT were unchanged in the mutants generated in the course of this work (see Supplemental Figure 2 online). It has to be mentioned that the activities for both enzymes are severalfold in excess compared with uptake rates of uridine (Wormit et al, 2004;Traub et al, 2007). However, it cannot be excluded that the cosubstrate of UPRT phosphoribosyl pyrophosphate limits uracil salvage as mutants in phosphoribosyl pyrophosphate synthesis showed that this metabolite may colimit growth rates in Arabidopsis (Koslowsky et al, 2008).…”

AtAzg1 and AtAzg2 comprise a novel family of purine transporters in Arabidopsis

“…None of the 12 identified Arabidopsis NAT loci are yet functionally characterized. Equilibrative nucleoside transporters (ENT) found in plants, protozoans and mammals, act primarily as nucleoside transporters but can also transport nucleobases adenine, guanine, xanthine and hypoxanthine or cytokinin [7][8][9][10][11][12][13]. Eight identified ENT loci are present in the Arabidopsis genome.…”

Identification of AtENT3 as the main transporter for uridine uptake in Arabidopsis roots

“…Recombinant AtENT1 expressed in yeast cells has been found to transport adenosine, guanosine, and cytidine in a pH-dependent manner [10,18]. However, it has been suggested that AtENT1 may not transport uridine based on competition assay [10], although uridine is efficiently transported by most ENT proteins characterized to date [19,20].…”

“…However, it has been suggested that AtENT1 may not transport uridine based on competition assay [10], although uridine is efficiently transported by most ENT proteins characterized to date [19,20]. The recombinant proteins of AtENTs 3, 4, 6, and 7 have all been found to transport adenosine, guanosine, cytidine, and uridine in a facilitative manner [17,18]. In contrast, the yeast-expressed AtENT8 is unable to mediate the uptake of any of the four nucleosides (adenosine, guanosine, cytidine, and uridine) tested so far [18].…”

“…The recombinant proteins of AtENTs 3, 4, 6, and 7 have all been found to transport adenosine, guanosine, cytidine, and uridine in a facilitative manner [17,18]. In contrast, the yeast-expressed AtENT8 is unable to mediate the uptake of any of the four nucleosides (adenosine, guanosine, cytidine, and uridine) tested so far [18]. Compared to AtENTs 1, 3, 4, 6, 7, and 8, there is still no information on the transport function of recombinant AtENT2.…”