Chimeric purine transporters of Aspergillus nidulans define a domain critical for function and specificity conserved in bacterial, plant and metazoan homologues

Abstract

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 highaffinity, 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.

Role of Intramembrane Polar Residues in the YgfO Xanthine Permease

“…In summary, His-31 (TM1) and Asn-93 (TM3) are crucial for affinity and/or specificity against purine analogues, whereas Glu-272 (TM8) and Asp-304 (TM9a) are irreplaceable for active xanthine transport. Taken together with our previous findings on the irreplaceable role of Gln-324 and Asn-325 in the NAT motif sequence (9) and on Asn-430 in TM12 being in the vicinity of the purine binding site (10), these data suggest that polar residues critical for the mechanism of YgfO cluster in TMs 1,3,8,9a, and 12 and the NAT motif sequence (Fig. 1).…”

“…It is important to note that Asp-304 of YgfO is at an amphipathic, putative transmembrane segment (TM9a) preceding the conserved NAT signature motif (residues 323-333). We have previously shown that the motif region contains two functionally irreplaceable residues, Gln-324, which is essential for high affinity binding and uptake, and Asn-325, which also interferes with substrate binding, 3 as well as residues important for substrate analogue selectivity that fall on the NEM-sensitive ␣-helical face of TM9b (Fig. 1) (9).…”

“…More than 1000 sequence entries are known, but few have been functionally characterized to date. The best studied eukaryotic member is UapA, a high affinity uric acid/xanthine:H ϩ symporter from the ascomycote Aspergillus nidulans (3)(4)(5)(6)(7). Studies with chimeric transporter constructs (3), site-directed mutagenesis, second-site suppressors, and kinetic inhibition analysis of ligand specificity have shown that a conserved NAT/NCS2 motif region between putative transmembrane helices 8 and 9 of UapA includes determinants of substrate recognition and selectivity, with at least one residue (Gln-408) implicated in binding with the imidazole moiety of purine (4), whereas a conserved QH motif at the middle of TM1 is important for activity and/or correct targeting to the plasma membrane (5), and an aromatic residue at the middle of TM12 (Phe-528) may act as a purine substrate selectivity filter (6).…”

Cysteine-scanning Analysis of Helices TM8, TM9a, and TM9b and Intervening Loops in the YgfO Xanthine Permease

“…It is interesting to note that Thr-280 and, especially, Ala-279 display a high degree of conservation among members of the NAT/NCS2 family (5,10) and that Ala-363, the residue corresponding to Ala-279 in the fungal homolog UapA (5), has been suggested as critical for function in Aspergillus nidulans, based on the loss-of-function phenotype of mutant A363D (18). Although a more comprehensive mutagenesis study of Ala-363 is missing (5,6), genetic analysis has shown that presence of a Gly in place of Ala-363 is compatible with full UapA activity (18), which is reminiscent of the wild-type expression and activity seen with mutant A279G in YgfO (Fig. 6).…”

“…The whole sequence region between TM8 and TM9b has been defined as a domain critical for function and specificity of the fungal UapA (5, 18), based initially on in vivo complementation of a set of chimeric transporter constructs in A. nidulans (18); this region appears to be involved in dynamic functional interactions with other parts of the protein (TM1, TM12) controlling affinity and specificity for substrate (5). Subsequent studies in both the fungal homolog UapA (4, 5) and the bacterial homolog YgfO (8 -11) established that a conserved NAT signature motif within this region (Fig.…”