Amino acid residues N-450 and Q(449) are critical for the uptake capacity and specificity of UapA, a prototype of a nucleobase-ascorbate transporter family

Specific carrier-mediated transport of purine and pyrimidine nucleobases ac ross cell membranes is a basic biological process in both prokaryotes and e ukaryotes. Recent in silico analysis has shown that the Aspergillus nidulan s (UapA, UapC) and bacterial (PbuX, UraA, PyrP) nucleobase transporters, an d a group of mammalian L-ascorbic acid transporters (SVCT1 and SVCT2), cons titute a unique protein family which includes putative homologues from arch ea, bacteria, plants and metazoans. The construction and functional analysi s of chimeric purine transporters (UapA-UapC) and UapA-specific missense mu tations in A. nidulans has previously shown that the region including amino acid residues 378-446 in UapA is critical for purine recognition and trans port. Here, we extend our studies on UapA structure-function relationships by studying missense mutations constructed within a 'signature' sequence mo tif [(F/Y/S)X(Q/E/P)NXGXXXXT(K/R/G)] which is conserved in the putative fun ctional region of all members of the nucleobase/ascorbate transporter famil y. Residues Q(449) and N-450 were found to be critical for purine recogniti on and transport. The results suggest that these residues might directly or indirectly be involved in specific interactions with the purine ring. In p articular, interaction of residue 449 with C-2 groups of purines might act as a critical molecular filter involved in the selection of transported sub strates. The present and previous mutagenic analyses in UapA suggest that s pecific polar or charged amino acid residues on either side of an amphipath ic alpha-helical transmembrane segment are critical for purine binding and transport.