Substitution F569S converts UapA, a specific uric acid-xanthine transporter, into a broad specificity transporter for purine-related solutes.
UapA, a highly specific uric acid-xanthine transporter in Aspergillus nidulans, is a member of a large family of nucleobase-ascorbate transporters conserved in all domains of life. We have investigated structure-function relationships in UapA, by studying chimeric transporters and missense mutations, and showed that specific polar or charged amino acid residues (E412, E414, Q449, N450, T457) on either side of an amphipathic alpha-helical transmembrane segment (TMS10) are critical for purine binding and transport. Here, the mutant Q449E, having no uric acid-xanthine transport activity at 25 degrees C, was used to isolate second-site revertants that restore function. Seven of them were found to have acquired the capacity to transport novel substrates (hypoxanthine and adenine) in addition to uric acid and xanthine. All seven revertants were found to carry the mutation F569S within the last transmembrane segment (TMS14) of UapA. Further kinetic analysis of a selected suppressor showed that UapA-Q449E/F569S transports with high affinity (K(M) values of 4-10 microM) xanthine, hypoxanthine and uracil. Uptake competition experiments suggested that UapA-Q449E/F569S also binds guanine, 6-thioguanine, adenosine or ascorbic acid. A strain carrying mutation F569S by itself conserves high-capacity, high-affinity (K(M) values of 1.5-15 microM), transport activity for purine-uracil transport. Compared to UapA-Q449E/F569S, UapA-F569S has a distinct capacity to bind several nucleobase-related compounds and different kinetic parameters of transport. These results show that molecular determinants external to the central functional domain (L9-TMS10-L10) are critical for the uptake specificity and transport kinetics of UapA.
Amillis
S
S
Faculty of Biology, Department of Botany, University of Athens, Panepistimioupolis, Athens, 15781, Greece.
Koukaki
M
M
Diallinas
G
G
D000225:Adenine / Q000378:metabolism; D000483:Alleles; D019943:Amino Acid Substitution / Q000235:genetics*; D001233:Aspergillus nidulans / Q000737:chemistry / Q000254:growth & development / Q000378:metabolism*; D001667:Binding, Competitive; D001692:Biological Transport; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal / Q000235:genetics; D019271:Hypoxanthine / Q000378:metabolism; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011687:Purines / Q000378:metabolism*; D012995:Solubility; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013489:Suppression, Genetic / Q000235:genetics; D014498:Uracil / Q000378:metabolism; D014508:Urea / Q000378:metabolism; D014527:Uric Acid / Q000378:metabolism; D019820:Xanthine / Q000378:metabolism
D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D011687:Purines; C083852:UAPA protein, Aspergillus nidulans; D019820:Xanthine; D014527:Uric Acid; D019271:Hypoxanthine; D014498:Uracil; D014508:Urea; D000225:Adenine