CHIMERIC PURINE TRANSPORTERS OF ASPERGILLUS-NIDULANS DEFINE A DOMAIN CRITICAL FOR FUNCTION AND SPECIFICITY CONSERVED IN BACTERIAL, PLANT AND METAZOAN HOMOLOGS

In Aspergillus nidulans, purine uptake is mediated by three transporte r proteins: UapA, UapC and AzgA, UapA and UapC have partially overlapp ing functions, are 62% identical and have nearly identical predicted t opologies, Their structural similarity is associated with overlapping substrate specificities; UapA is a high-affinity, high-capacity specif ic xanthine/uric acid transporter. UapC is a low/moderate-capacity gen eral purine transporter. We constructed and characterized UapA/ UapC, UapC/UapA and UapA/UapC/UapA chimeric proteins and UapA point mutation s. The region including residues 378-446 in UapA (336-404 in UapC) has been shown to be critical for purine recognition and transport. Withi n this region, we identified: (i) one amino acid residue (A404) import ant 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 t o man. The UapC/UapA series of chimeras behaves in a linear pattern an d leads to an univocal assignment of functional domains while the anal ysis of the reciprocal and 'sandwich' chimeras revealed unexpected int er-domain interactions, cDNAs coding for transporters including the sp ecificity region defined by these studies have been identified for the first time in the human and Caenorhabditis elegans databases.