title: Mutational analysis and modeling reveal functionally critical residues in transmembrane segments 1 and 3 of the UapA transporter.
abstract: Earlier, we identified mutations in the first transmembrane segment (TMS1) of UapA, a uric acid-xanthine transporter in Aspergillus nidulans, that affect its turnover and subcellular localization. Here, we use one of these mutations (H86D) and a novel mutation (I74D) as well as genetic suppressors of them, to show that TMS1 is a key domain for proper folding, trafficking and turnover. Kinetic analysis of mutants further revealed that partial misfolding and deficient trafficking of UapA does not affect its affinity for xanthine transport, but reduces that of uric acid and confers a degree of promiscuity towards the binding of other purines. This result strengthens the idea that subtle interactions among domains not directly involved in substrate binding refine the selectivity of UapA. Characterization of second-site suppressors of H86D revealed a genetic interaction of TMS1 with TMS3, the latter segment shown for the first time to be important for UapA function. Systematic mutational analysis of polar and conserved residues in TMS3 showed that Ser154 is crucial for UapA transport activity. Our results are in agreement with a topological model of UapA built on the recently published structure of UraA, a bacterial homolog of UapA.
authors: Department of Botany, Faculty of Biology, University of Athens, Panepistimioupolis, Athens 15781, Greece.
authors: Sotiris
authors: S
authors: Amillis
authors: Vasiliki
authors: V
authors: Kosti
authors: Areti
authors: A
authors: Pantazopoulou
authors: Emmanuel
authors: E
authors: Mikros
authors: George
authors: G
authors: Diallinas
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001233:Aspergillus nidulans / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D033702:Nucleobase Transport Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport
chemical_list: D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D033702:Nucleobase Transport Proteins; C083852:UAPA protein, Aspergillus nidulans