Nucleobase-ascorbate Transporter Research Articles

Specific Interdomain Synergy in the UapA Transporter Determines Its Unique Specificity for Uric Acid among NAT Carriers

UapA, a uric acid–xanthine permease of Aspergillus nidulans, has been used as a prototype to study structure–function relationships in the ubiquitous nucleobase–ascorbate transporter (NAT) family. Using novel genetic screens, rational mutational design, chimeric NAT molecules, and extensive transport kinetic analyses, we show that dynamic synergy between three distinct domains, transmembrane segment (TMS)1, the TMS8–9 loop, and TMS12, defines the function and specificity of UapA. The TMS8–9 loop includes four residues absolutely essential for substrate binding and transport (Glu356, Asp388, Gln408, and Asn409), whereas TMS1 and TMS12 seem to control, through steric hindrance or electrostatic repulsion, the differential access of purines to the TMS8–9 domain. Thus, UapA specificity is determined directly by the specific interactions of a given substrate with the TMS8–9 loop and indirectly by interactions of this loop with TMS1 and TMS12. We finally show that intramolecular synergy among UapA domains is highly specific and propose that it forms the basis for the evolution of the unique specificity of UapA for uric acid, a property not present in other NAT members.

Cysteine-scanning Analysis of Putative Helix XII in the YgfO Xanthine Permease: ILE-432 AND ASN-430 ARE IMPORTANT

Transmembrane helix XII of UapA, the major fungal homolog of the nucleobase-ascorbate transporter (NAT/NCS2) family, has been proposed to contain an aromatic residue acting as a purine-selectivity filter, distinct from the binding site. To analyze the role of helix XII more systematically, we employed Cys-scanning mutagenesis of the Escherichia coli xanthine-specific homolog YgfO. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequence 419ILPASIYVLVENPICAGGLTAILLNIILPGGY450 (the putative helix XII is underlined) was replaced individually with Cys. Of the 32 single-Cys mutants, 25 accumulate xanthine to 80-130% of the steady state observed with C-less YgfO, six (P421C, S423C, I424C, Y425C, L427C, G436C) accumulate to low levels (15-40%), and I432C is inactive. Immunoblot analysis shows that P421C and I432C display low expression in the membrane. Extensive mutagenesis reveals that replacement of Ile-432 with equally or more bulky side chains abolishes active transport without affecting expression, whereas replacement with smaller side chains allows activity but impairs affinity for the analogues 1-methyl and 6-thioxanthine. Only three of the single-Cys mutants of helix XII (V426C, N430C, and N443C) are sensitive to inactivation by N-ethylmaleimide. N430C is highly sensitive, with an IC50 of 10 microm, and is completely protected against inactivation in the presence of 2-thioxanthine, a high affinity substrate analogue. Other xanthine analogues are poorly bound by N430C, whereas replacement of Asn-430 with Thr inactivates the permease. The findings suggest that Ile-432 and Asn-430 of helix XII are crucial for purine uptake and affinity, and Asn-430 is probably at the vicinity of the binding site.

The nucleobase–ascorbate transporter (NAT) family: genomics, evolution, structure–function relationships and physiological role

This review summarizes knowledge concerning a ubiquitous plasma transmembrane protein family that mediates nucleobase or ascorbate secondary active transport (NAT). We show that prototype bacterial and mostly fungal members have become unique model systems to unravel structure-function relationships and regulation of expression, using classical and reverse genetics, as well as biochemical approaches. We discuss the importance of NAT-mediated ascorbate transport in mammals and how changes in substrate specificity, from different nucleobases to ascorbate, might have evolved at the molecular level. Finally, we also discuss how modelling NAT-purine interactions might constitute a step towards the use of NAT proteins as specific gateways for targeting pathogenic microbes.

Cysteine-scanning Analysis of the Nucleobase-Ascorbate Transporter Signature Motif in YgfO Permease of Escherichia coli: Gln-324 AND Asn-325 ARE ESSENTIAL, AND Ile-329–Val-339 FORM AN α-HELIX

The nucleobase-ascorbate transporter (NAT) signature motif is a conserved sequence motif of the ubiquitous NAT/NCS2 family implicated in defining the function and selectivity of purine translocation pathway in the major fungal homolog UapA. To analyze the role of NAT motif more systematically, we employed Cys-scanning mutagenesis of the Escherichia coli xanthine-specific homolog YgfO. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequence (315)GSIPITTFAQNNGVIQMTGVASRYVG(340) (motif underlined) was replaced individually with Cys. Of the 26 single-Cys mutants, 16 accumulate xanthine to > or =50% of the steady state observed with C-less YgfO, 4 accumulate to low levels (10-25% of C-less), F322C, N325C, and N326C accumulate marginally (5-8% of C-less), and P318C, Q324C, and G340C are inactive. When transferred to wild type, F322C(wt) and N326C(wt) are highly active, but P318G(wt), Q324C(wt), N325C(wt), and G340C(wt) are inactive, and G340A(wt) displays low activity. Immunoblot analysis shows that replacements at Pro-318 or Gly-340 are associated with low or negligible expression in the membrane. More extensive mutagenesis reveals that Gln-324 is critical for high affinity uptake and ligand recognition, and Asn-325 is irreplaceable for active xanthine transport, whereas Thr-332 and Gly-333 are important determinants of ligand specificity. All single-Cys mutants react with N-ethylmaleimide, but regarding sensitivity to inactivation, they fall to three regions; positions 315-322 are insensitive to N-ethylmaleimide, with IC(50) values > or =0.4 mM, positions 323-329 are highly sensitive, with IC(50) values of 15-80 microM, and sensitivity of positions 330-340 follows a periodicity, with mutants sensitive to inactivation clustering on one face of an alpha-helix.

Identification and Expression Analysis of Twelve Members of the Nucleobase–Ascorbate Transporter (NAT) Gene Family in Arabidopsis thaliana

By screening genome databases, 12 genes encoding membrane proteins homologous to nucleobase-ascorbate transporters (NATs) were identified in Arabidopsis thaliana. A similar number of genes was found in the rice genome. The plant NAT proteins split into five clades (I-V) based on protein multisequence alignments. This classification nicely correlates with the patterns of organ- and tissue-specific expression during the whole life cycle of A. thaliana. Interestingly, expression of two members of clade III, AtNAT7 and AtNAT8, was found to be up-regulated in undifferentiated tissues such as callus or tumors produced by Agrobacterium tumefaciens. Clade V comprises AtNAT12 possessing a hydrophilic N-terminal extension. Transient expression of green fluorescent protein (GFP) fusions in different systems showed that AtNAT12 along with AtNAT7 and -8 are located in the plasma membrane. Mutations in any of the AtNAT genes do not induce phenotypic alterations. The absence of obvious mutant phenotypes in single but also in double and triple mutants suggests a high degree of functional redundancy between AtNAT genes, but might also point to redundant functions provided by genes or pathways unrelated to the AtNATs.

A Novel-type Substrate-selectivity Filter and ER-exit Determinants in the UapA Purine Transporter

We present a functional analysis of the last alpha-helical transmembrane segment (TMS12) of UapA, a uric acid-xanthine/H+ symporter in Aspergillus nidulans and member of the nucleobase-ascorbate transporter (NAT) family. First, we performed a systematic mutational analysis of residue F528, located in the middle of TMS12, which was known to be critical for UapA specificity. Substitution of F528 with non-aromatic amino acid residues (Ala, Thr, Ser, Gln, Asn) did not affect significantly the kinetics of UapA for its physiological substrates, but allowed high-capacity transport of several novel purines and pyrimidines. Allele-specific combinations of F528 substitutions with mutations in Q408, a residue involved in purine binding, led to an array of UapA molecules with different kinetic and specificity profiles. We propose that F528 plays the role of a novel-type selectivity filter, which, in conjunction with a distinct purine-binding site, control UapA-mediated substrate translocation. We further studied the role of TMS12 by analysing the effect of its precise deletion and chimeric molecules in which TMS12 was substituted with analogous domains from other NATs. The presence of any of the TMS12 tested was necessary for ER-exit while their specific amino acid composition affected the kinetics of chimeras.

Comparative substrate recognition by bacterial and fungal purine transporters of the NAT/NCS2 family

We compared the interactions of purines and purine analogues with representative fungal and bacterial members of the widespread Nucleobase-Ascorbate Transporter (NAT) family. These are: UapA, a well-studied xanthine-uric acid transporter of A. nidulans, Xut1, a novel transporter from C. albicans, described for the first time in this work, and YgfO, a recently characterized xanthine transporter from E. coli. Using transport inhibition experiments with 64 different purines and purine-related analogues, we describe a kinetic approach to build models on how NAT proteins interact with their substrates. UapA, Xut1 and YgfO appear to bind several substrates via interactions with both the pyrimidine and imidazol rings. Fungal homologues interact with the pyrimidine ring of xanthine and xanthine analogues via H-bonds, principally with N1-H and =O6, and to a lower extent with =O2. The E. coli homologue interacts principally with N3-H and =O2, and less strongly with N1-H and =O6. The basic interaction with the imidazol ring appears to be via a H-bond with N9. Interestingly, while all three homologues recognize xanthines with similar high affinities, interaction with uric acid or/and oxypurinol is transporter-specific. UapA recognizes uric acid with high affinity, principally via three H-bonds with =O2, =O6 and =O8. Xut1 has a 13-fold reduced affinity for uric acid, based on a different set of interactions involving =O8, and probably H atoms from positions N1, N3, N7 or N9. YgfO does not recognize uric acid at all. Both Xut1 and UapA recognize oxypurinol, but use different interactions reflected in a nearly 26-fold difference in their affinities for this drug, while YgfO interacts with this analogue very inefficiently.

Arabidopsis thaliana locus At5g62890, a nucleobase-ascorbate transporter family member, is preferentially expressed in carpel transmitting tract and tapetal cells

The expression pattern for Arabidopsis locus At5g62890 (MQB2.190) is investigated at the molecular and whole plant level. MQB2.190 belongs to the nucleobase-ascorbate transporter (NAT) family which includes transporters of purines, pyrimidines and ascorbate. A full-length cDNA clone was sequenced verifying that MQB2.190 is actively expressed and encodes a NAT protein. Northern blot and RT-PCR analysis reveal that MQB2.190 is expressed at a low level in a variety of tissues, but is preferentially expressed in flowers. Whole plant gene expression was investigated by generating transgneic Arabidopsis with the MQB2.190 promoter driving GUS gene. Visible GUS staining was detected in the central tissue of carpels and siliques and faintly in anthers, but was absent in young seedlings, roots, leaves, stems or other floral tissues. MQB2.190 transcripts were precisely localized to the central transmitting tract of the carpel and in the tapetal cell layer in anthers using RNA in situ hybridization.

Substitution F569S converts UapA, a specific uric acid-xanthine transporter, into a broad specificity transporter for purine-related solutes

UapA, a highly specific uric acid-xanthine transporter in Aspergillus nidulans, is a member of a large family of nucleobase-ascorbate transporters conserved in all domains of life. We have investigated structure-function relationships in UapA, by studying chimeric transporters and missense mutations, and showed that specific polar or charged amino acid residues (E412, E414, Q449, N450, T457) on either side of an amphipathic α-helical transmembrane segment (TMS10) are critical for purine binding and transport. Here, the mutant Q449E, having no uric acid-xanthine transport activity at 25°C, was used to isolate second-site revertants that restore function. Seven of them were found to have acquired the capacity to transport novel substrates (hypoxanthine and adenine) in addition to uric acid and xanthine. All seven revertants were found to carry the mutation F569S within the last transmembrane segment (TMS14) of UapA. Further kinetic analysis of a selected suppressor showed that UapA-Q449E/F569S transports with high affinity ( K M values of 4-10 μM) xanthine, hypoxanthine and uracil. Uptake competition experiments suggested that UapA-Q449E/F569S also binds guanine, 6-thioguanine, adenosine or ascorbic acid. A strain carrying mutation F569S by itself conserves high-capacity, high-affinity ( K M values of 1.5-15 μM), transport activity for purine-uracil transport. Compared to UapA-Q449E/F569S, UapA-F569S has a distinct capacity to bind several nucleobase-related compounds and different kinetic parameters of transport. These results show that molecular determinants external to the central functional domain (L9-TMS10-L10) are critical for the uptake specificity and transport kinetics of UapA.

Amino acid residues N450 and Q449 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 across cell membranes is a basic biological process in both prokaryotes and eukaryotes. Recent in silico analysis has shown that the Aspergillus nidulans (UapA, UapC) and bacterial (PbuX, UraA, PyrP) nucleobase transporters, and a group of mammalian L-ascorbic acid transporters (SVCT1 and SVCT2), constitute a unique protein family which includes putative homologues from archea, bacteria, plants and metazoans. The construction and functional analysis of chimeric purine transporters (UapA-UapC) and UapA-specific missense mutations in A. nidulans has previously shown that the region including amino acid residues 378-446 in UapA is critical for purine recognition and transport. Here, we extend our studies on UapA structure-function relationships by studying missense mutations constructed within a 'signature' sequence motif [(F/Y/S)X(Q/E/P)NXGXXXXT(K/R/G)] which is conserved in the putative functional region of all members of the nucleobase/ascorbate transporter family. Residues Q449 and N450 were found to be critical for purine recognition and transport. The results suggest that these residues might directly or indirectly be involved in specific interactions with the purine ring. In particular, interaction of residue 449 with C-2 groups of purines might act as a critical molecular filter involved in the selection of transported substrates. The present and previous mutagenic analyses in UapA suggest that specific polar or charged amino acid residues on either side of an amphipathic alpha-helical transmembrane segment are critical for purine binding and transport.