This review summarizes knowledge concerning a ubiquitous plasma transmembrane protein family that mediates nucleobase or ascorbate secondary active transport (NAT). We show that prototype bacterial and mostly fungal members have become unique model systems to unravel structure-function relationships and regulation of expression, using classical and reverse genetics, as well as biochemical approaches. We discuss the importance of NAT-mediated ascorbate transport in mammals and how changes in substrate specificity, from different nucleobases to ascorbate, might have evolved at the molecular level. Finally, we also discuss how modelling NAT-purine interactions might constitute a step towards the use of NAT proteins as specific gateways for targeting pathogenic microbes.
UapA, a uric acid-xanthine permease of Aspergillus nidulans, has been used as a prototype to study structure-function relationships in the ubiquitous nucleobase-ascorbate transporter (NAT) family. Using novel genetic screens, rational mutational design, chimeric NAT molecules, and extensive transport kinetic analyses, we show that dynamic synergy between three distinct domains, transmembrane segment (TMS)1, the TMS8-9 loop, and TMS12, defines the function and specificity of UapA. The TMS8-9 loop includes four residues absolutely essential for substrate binding and transport (Glu356, Asp388, Gln408, and Asn409), whereas TMS1 and TMS12 seem to control, through steric hindrance or electrostatic repulsion, the differential access of purines to the TMS8-9 domain. Thus, UapA specificity is determined directly by the specific interactions of a given substrate with the TMS8-9 loop and indirectly by interactions of this loop with TMS1 and TMS12. We finally show that intramolecular synergy among UapA domains is highly specific and propose that it forms the basis for the evolution of the unique specificity of UapA for uric acid, a property not present in other NAT members.
We cloned and characterized an Aspergillus nidulans gene, called fcyB, encoding the closest homologue to the yeast Fcy2p/Fcy21p permeases. Deletion of fcyB (DeltafcyB) does not affect growth, development, reproduction or bulk purine uptake, but eliminates the leaky growth on purines of DeltaazgADeltauapCDeltauapA strains, lacking all known purine transporters, and confers resistance to the antifungal 5-fluorocytosine. Kinetic analyses showed FcyB is a low-capacity, high-affinity, cytosine-purine transporter sharing similar molecular interactions for substrate recognition with the yeast Fcy2p/Fcy21p carriers. fcyB transcription is highly activated during germination but drops at low constitutive levels throughout vegetative development. UaY-mediated purine induction of fcyB transcription is only moderate, while ammonium represses transcription through an AreA-dependent mechanism. A strain expressing FcyB-GFP confirms a low protein expression level in the plasma membrane of vegetative mycelia, but reveals an abundant expression in sexual and asexual compartments. FcyB-GFP was also shown to be downregulated by endocytosis in response to ammonia or the presence of cytosine. The expression profile of FcyB supports that its main physiological role is cytosine-purine scavenging.