Chimeric purine transporters of Aspergillus nidulans define a domain critical for function and specificity conserved in bacterial, plant and metazoan homologues.
In Aspergillus nidulans, purine uptake is mediated by three transporter proteins: UapA, UapC and AzgA. UapA and UapC have partially overlapping functions, are 62% identical and have nearly identical predicted topologies. Their structural similarity is associated with overlapping substrate specificities; UapA is a high-affinity, high-capacity specific xanthine/uric acid transporter. UapC is a low/moderate-capacity general purine transporter. We constructed and characterized UapA/UapC, UapC/UapA and UapA/UapC/UapA chimeric proteins and UapA point mutations. The region including residues 378-446 in UapA (336-404 in UapC) has been shown to be critical for purine recognition and transport. Within this region, we identified: (i) one amino acid residue (A404) important for transporter function but probably not for specificity and two residues (E412 and R414) important for UapA function and specificity; and (ii) a sequence, (F/Y/S)X(Q/E/P) NXGXXXXT(K/R/G), which is highly conserved in all homologues of nucleobase transporters from bacteria to man. The UapC/UapA series of chimeras behaves in a linear pattern and leads to an univocal assignment of functional domains while the analysis of the reciprocal and 'sandwich' chimeras revealed unexpected inter-domain interactions. cDNAs coding for transporters including the specificity region defined by these studies have been identified for the first time in the human and Caenorhabditis elegans databases.
Diallinas
G
G
Institute of Molecular Biology and Biotechnology, FORTH, P.O. Box 1527, Heraklion 71110, Crete.
Valdez
J
J
Sophianopoulou
V
V
Rosa
A
A
Scazzocchio
C
C
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000502:physiology; D000818:Animals; D001233:Aspergillus nidulans / Q000201:enzymology*; D001665:Binding Sites; D001692:Biological Transport; D017124:Conserved Sequence / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D005656:Fungal Proteins; D006801:Humans; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017354:Point Mutation; D011687:Purines / Q000378:metabolism; D011688:Purinones / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000596:Amino Acids; D018076:DNA, Complementary; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D011687:Purines; D011688:Purinones; D011993:Recombinant Fusion Proteins; C083852:UAPA protein, Aspergillus nidulans; C114088:UapC protein, Aspergillus nidulans