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.
Institute of Molecular Biology and Biotechnology, FORTH, P.O. Box 1527, Heraklion 71110, Crete.
G
G
Diallinas
J
J
Valdez
V
V
Sophianopoulou
A
A
Rosa
C
C
Scazzocchio
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