title: Transport assays in filamentous fungi: kinetic characterization of the UapC purine transporter of Aspergillus nidulans.
abstract: Transport assays allow the direct kinetic analysis of a specific transporter by measuring apparent Km and Vmax values, and permit the characterization of substrate specificity profiles through competition assays. In this protocol we describe a rapid and easy method for performing uptake assays in the model filamentous ascomycete Aspergillus nidulans. Our method makes use of A. nidulans germinating conidiospores at a defined morphological stage in which most transporters show maximal expression, avoiding technical difficulties associated with the use of mycelia. In combination with the ease of construction of genetic null mutants in A. nidulans, our method allows the rigorous characterization of any transporter in genetic backgrounds that are devoid of other transporters of similar specificity. Here, we use this method to characterize the kinetic parameters and the specificity profile of UapC, a uric acid-xanthine transporter present in all ascomycetes and member of the ubiquitous Nucleobase-Ascorbate Transporter family, in specific genetic backgrounds lacking other relevant transporters.
authors.affiliation: Faculty of Biology, University of Athens, Panepistimioupolis, Athens 15784, Greece.
authors.forename: Emilia
authors.initials: E
authors.lastname: Krypotou
authors.affiliation: Faculty of Biology, University of Athens, Panepistimioupolis, Athens 15784, Greece. Electronic address: diallina@biol.uoa.gr.
authors.forename: George
authors.initials: G
authors.lastname: Diallinas
mesh_terms: D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000378:metabolism*; D001692:Biological Transport; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010117:Oxypurinol / Q000378:metabolism; D011687:Purines / Q000378:metabolism*; D014527:Uric Acid / Q000378:metabolism; D019820:Xanthine / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D011687:Purines; C083852:UAPA protein, Aspergillus nidulans; C114088:UapC protein, Aspergillus nidulans; D019820:Xanthine; D014527:Uric Acid; D010117:Oxypurinol; C030985:purine
keywords: Germinating conidiospores; NAT; Specificity; Uptake; Uric acid; Xanthine